As is well appreciated, Agrobacterium tumefaciens is a useful bacteria for delivering and integrating desired nucleic acids, such as desirable transgenes, into a plant cell genome. The bacterium's transfer DNA (T-DNA) is the vehicle that shuttles the transgene from the tumor-inducing plasmid contained within the bacterium strain and into the plant's genetic material. The agricultural industry exploits this capability in order to genetically modify crops to express desirable genes and traits.
Typically, a selectable marker gene is positioned alongside the transgene in the T-DNA, so that it is integrated into the plant genome along with the transgene. The expression of the marker helps to identify those cells that contain an integrated transgene. Some common markers include bacterial antibiotic resistance genes, such as neomycin phosphotransferase, “nptII” (Fraley et al., Proc. Natl. Acad. Sci. 80:4803-4807, 1983) and hygromycin phosphotransferase, “hpt” (Waldron et al., Plant Mol. Biol. 5:103-108, 1985). Other bacterial fungal markers include the fungal mycotoxin resistance gene, cyanamide hydratase, “cah” (Weeks et al., Crop Sci. 40:1749-1754, 2000), phosphomannose isomerase, “manA” or “pmi” (Joersbo et al., Physiol. Plant 105:109-115, 1999) and threonine deaminase, “TD” (Ebmeier et al., Planta 218:751-758, 2004).
It is possible to co-assign a marker to a specific gene and thereby devise a T-DNA, which contains multiple genes and multiple markers for ultimate integration into a plant genome. Thus, this so-called “gene stacking” strategy combines several desired traits into one plant line. The ability to stack numerous genes, however, depends on the availability and appropriateness of the co-assigned markers. Hence, gene stacking can be limited by its dependence on marker genes.
Another concern, is that some believe markers pose potential risks to human and animal health and to the environment. For instance, there is concern that antibiotic- and herbicide-resistance genes may escape from the plants into which they are engineered and into the environment. Thus, those resistance genes, outside of the modified plant's genome, may confer an adaptive advantage to weeds and pathogens that take them into their own genomes (Dale et al., Nat. Biotech. 20:567-574, 2002).
Accordingly, several strategies have been devised to excise a selectable marker from a transgenic plant. One such method employs site-specific recombinase enzymes to remove the marker from its locus in the plant genome. See Dale and Ow, Proc. Natl. Acad. Sci. 23:10558-10562, 1991; Kilby et al., Plant J. 8:637-652, 1994; and Sugita et al., Plant J. 22:461-469, 1999. The removal success rate, however, is quite low. Furthermore, the excision process typically leaves foreign DNA elements embedded in the plant genome.
Another method involves elimination of markers from the plant genome by co-transformation. This strategy essentially entails delivering and integrating the gene of interest and its marker into the plant genome using two T-DNAs. The idea is that it is easier to cross-out the marker from the plant line, while retaining the desired gene, because the gene and marker are not linked genetically. Hence, two distinct integration events must occur thereby permitting the distinct marker T-DNA element to be backcrossed out or otherwise segregated from the distinct gene because they exist at unlinked locations.
The T-DNAs that contain the marker and the gene may exist within the same binary vector or on different binaries. Similarly, a T-DNA containing a marker may be on a plasmid that is contained within an Agrobacterium strain that is the same or different as the strain containing the plasmid with the desired gene or in the backbone of a single binary vector. See Puchta, Plant Cell Tiss. Organ Cult., 74:123-134, 2003, and Huang et al., Transgen. Res. 13:451-461, 2004.
Unfortunately, these marker-removal strategies are time-consuming and can be financially draining, which is problematic because it is the absence of marker genes that helps to lower the cost of de-regulation and facilitate commercialization of genetically modified crops (Smyth et al., Nat. Biotech 20:537-541, 2002). More importantly, these strategies are useless in crops that are sterile and they cannot be applied to clonally-propagated crops such as potato.
Yet an added concern is not only the removal of the marker gene but the fact that Agrobacterium-mediated transformation more often than not introduces unnecessary plasmid backbone DNA sequences into the plant genome. That is, the enzymes that splice the T-DNA from the plasmid vector do not always cut the plasmid precisely. For example, the enzymes may cut the plasmid beyond the ends of the T-DNA, which are delineated by left and right border sequences that serve as recognition cleavage sites for those enzymes. Hence, the T-DNA, once spliced from the plasmid, may be longer than expected and thereby carry plasmid backbone sequences in addition to the marker and gene of interest.
These backbone sequences also can integrate into the plant genome. Indeed, 75% of all integration events contain backbone elements in Solanaceous species (Kononov et al., Plant J. 11:945-957, 1997; Heeres et al., Euphytica 124:13-22, 2002; Rommens et al., Plant Physiol. 135:421-431, 2004).
This is an added concern because the backbone sequences of tumor-inducing plasmids that are used in Agrobacterium-mediated transformation contain many different genetic elements, such as origins of replication, bacterial selectable marker genes, and other foreign regulatory elements. Agricultural and environmental authorities often find the presence of such extraneous foreign DNA in a plant genome to be unacceptable, despite the benefits conferred by expression of the co-integrated gene of interest.
Accordingly, in addition to devising strategies for removing markers, strategies also must be developed to identify those transgenic plants that also contain plasmid backbone sequences. These include polymerase chain reaction-based amplification and Southern blotting to identify the retention of known plasmid sequences in the plant's genetic makeup. Both screening methods are tedious and expensive, especially when backbone integration frequencies are high.
Placing lethal genes in the backbone to eliminate cells that are transformed with the backbone is potentially useful. See U.S. Pat. No. 6,521,458. Such genes, however, can cause problems to surrounding cells, such as impairing their ability to regenerate.
The conditionally-lethal gene cytosine deaminase, (codA), converts non-toxic 5-fluorocytosine (5-FU) to toxic 5-fluorouracil. 5-FU is known to migrate, however, to cells that do not contain any plasmid backbone sequences. Moreover, the method of using conditionally-lethal genes require the addition of exogenous substrates, which means another step in medium preparation or explant transfer.
The use of non-lethal marker genes in the backbone has been suggested as an alternative to lethal, conditionally lethal or scorable genes, such as GUS, beta glucuronidase. See U.S. patent application publication U.S. 2004/0237142.
The non-lethal genes provide a visual means to distinguish transgenic events that contain the vector backbone. Such genes include any that are involved in plant hormone biosynthesis, plant hormone degradation, plant hormone signaling, or metabolic interference.
This method identifies the transgenic plants that contain plasmid DNA without doing PCR or Southern blot screening, but it requires the use of a positive selection marker within the T-DNA and, therefore, is not a marker-free method.
One method was developed that exploited the utility of the marker gene, while selecting against its integration. By employing positive selection for transient marker gene expression followed by negative selection against marker gene stable integration, roughly 25% of regenerated plants contain the transgene of interest but no marker gene. See Rommens et al., Plant Physiol. 135:421-431, 2004, which is incorporated herein by reference.
Another method employs “armed” Agrobacterium strains that still contain their own hormone genes (Mihalka et al., Plant Cell Rep. 21:778-784, 2003, Aronen et al. Plant Cell Tissue Organ Cult. 70:147-154, 2002). A gene of interest is engineered into a disarmed binary plasmid and introduced into special “shooter” mutants that contain a native, oncogenic (or “armed”) Ti plasmid. Although this method will generally provide marker-free shoots at low frequency, it requires the use of special oncogenic strains. Another problem is the probability of having an backbone integration event with the gene of interest is compounded by the use of two binaries rather than one.
The present invention details a new method that requires expression of a hormone synthesis gene or genes positioned within the backbone of a plasmid having a transfer DNA (P-DNA or T-DNA). The hormone synthesis gene drives regeneration of marker-free plants harboring the transfer DNA. Selection against integration of backbone sequences is accomplished at regeneration and therefore, simplifies the identification of events without superfluous DNA sequences.